Sonic hedgehog (Shh) signaling is critical for normal vertebrate development and adult health. Mutations that alter the activation of the Shh pathway lead to developmental abnormalities including polydactyly and holoprosencephaly. Derepression of Shh signaling in adults contributes to the progression of multiple cancers, including basal cell carcinoma. A key issue for understanding the consequences of active Shh signaling is whether downstream targets are regulated directly, or indirectly through secondary signaling intermediates. This proposal seeks to ask this question using the avian limb bud as a model system. The Laufer laboratory will generate replication-competent avian retroviruses that express Shh, a membrane tethered Shh:CD4 fusion protein, or mutant Smo proteins that constitutively activate the Shh pathway. Limbs will be infected with these viruses under various targeting regimes, and then subjected to whole mount in situ hybridization histochemistry to determine the conditions required for maximal regulation of various Shh targets. Targets include Shh pathway genes, BMP family signaling molecules and Hox transcription factors. Replication-defective viruses carrying the same genes will be used to infect limb buds under the predetermined conditions. Target gene expression will be analyzed in sectioned limb tissue simultaneously immunochemically stained to detect infect cells. Comparison of the spatial relationship between infected cells and target gene expression will allow determination of the cell-autonomy of each response. Possible temporal changes in the nature of each response will also be determined by varying the developmental stage of viral infection, or the duration of exposure to each signal.